TGF-β/Smad7信号通路调节溃疡性结肠炎肠黏膜通透性的分子机制研究

Ulcerative colitis (UC) is a nonspecific inflammatory disease characterized by a tendency toward relapse or recurrence. Improving intestinal mucosal permeability is benefit to reduce intestinal inflammation, promote mucosal healing and maintain remission. Recently，it has been demonstrated that MLCK plays a critical role in systole of cytokeleton in intestinal mucosal permeability by phosphorvlating MLC，and TGF-beta/Smad7 signaling pathway plays a important role in the persistent inflammation of colon. However，whether the TGF-beta/Smad7 signaling pathway could regulate the expression of MLCK and affect the intestinal mucosal permeability in colitis，remains unclear. Therefore, the colitis model in mice would be established in this study and intestinal mucosal permeability would be evaluated by methods of transmission electron microscope， immunohistochemistry and functional tests in vivo and vitro. The drugs such as anti-TNF-a antibody and 5-ASA would be administrated to prevent colitis in this study. Critical molecules such as MLCK and the TGF-beta/Smad7 signaling pathway would be analyzed by methods such as Western blot and RT-PCR. To thoroughly investigate the molecular mechanisms of TGF-β/Smad7 signaling pathway on intestinal mucosal permeability, the caco2 cell model would also be established and the inhibitors of Smad7 and MLCK would be administrated in the cellular experiment. The methods such as Western blot and RT-PCR would also be used to analyze the TGF-β/Smad7 signaling pathway. The study of molecular mechanisms of TGF-β/Smad7 signaling pathway on intestinal mucosal permeability may pave the way for studies of pathogenesis and new therapeutic approaches in UC.

溃疡性结肠炎是一种反复发作的非特异性炎性疾病，改善肠黏膜通透性，控制肠道炎症，可促进黏膜愈合和维持缓解，意义重大。近期发现，MLCK磷酸化引起细胞骨架收缩在肠黏膜通透性调控机制中具有关键作用。TGF-β/Smad7信号通路在结肠持续性炎症中起重要作用，但是否调控MLCK表达、影响结肠炎肠黏膜通透性尚不明确。故本研究拟在建立结肠炎小鼠模型基础上，采用透射电镜、免疫组化、体内体外通透性功能检测等方法评估结肠炎小鼠肠黏膜通透性的结构和功能改变，并给予抗TNF-a单抗和5-ASA干预，运用Western blot、RT-PCR等方法分析肠黏膜通透性改变的关键分子MLCK和TGF-β/Smad7信号通路。再建立细胞模型，给予Smad7和MLCK抑制剂，运用Western blot、RT-PCR等方法深入探讨TGF-β/Smad7信号通路对肠黏膜通透性的调节，为UC发病机制和新的治疗药物研究奠定基础。

溃疡性结肠炎（UC）是一种反复发作的非特异性炎性疾病，改善肠粘膜通透性，控制肠道炎症，可促进粘膜愈合和维持缓解，意义重大。近期研究发现，MLCK磷酸化引起细胞骨架收缩在肠粘膜通透性调控机制中具有关键作用。TGF-beta/Smad7信号通路在结肠持续性炎症中起重要作用，但是否调控MLCK表达、影响结肠炎肠粘膜通透性尚不明确。故本研究在建立DSS结肠炎小鼠模型基础上，采用透射电镜（TEM）、免疫组化、体内体外通透性功能检测等方法评估结肠炎小鼠肠粘膜通透性的结构和功能改变，结果发现结肠炎小鼠肠粘膜通透性增高，肠组织中EB含量和血FITC明显升高（P＜0.05 ），TEM检查发现小肠上皮细胞破损、细胞紧密连接减少；小肠MLCK蛋白表达增强、MLCK酶活性增高，而E-cadherin、ZO-1和occludin等紧密连接相关蛋白表达减少。给予抗TNF-a单抗和5-ASA干预，可改善结肠炎小鼠症状，降低DAI、HI评分，并且改善肠粘膜通透性。进一步运用Western blot、RT-PCR等方法分析发现抗TNF-a单抗和5-ASA组小鼠小肠上皮细胞MLCK、Smad7表达水平降低（P＜0.05），而TGF-beta表达水平增高（P＜0.05）。为进一步证实TGF-beta、Smad7信号通路对肠粘膜通透性的调节，建立了Caco2单层细胞模型，给予ML-7及不同剂量的Smad7抑制剂进行干预，采用Millipore电阻仪测定单层细胞TEER，采用EB和FITC检测细胞通透性，运用ELISA和免疫荧光、Western blot检测MLCK活性和蛋白表达，采用Western blot、RT-PCR等方法检测TGF-beta和Smad7表达。结果发现，药物干预组细胞通透性改善，MLCK活性和蛋白表达降低，Smad7蛋白表达水平降低（P＜0.05），而TGF-beta表达水平增高（P＜0.05）。研究证实提高TGF-beta表达水平或降低Smad7表达水平，MLCK表达降低，肠粘膜通透性改善。这一发现有助于UC发病机制和新的治疗药物研究。